1. Field of the Invention
The present invention relates to a method for identifying nucleic acid sequences using hybridization probes. More specifically, the present invention relates to a method for identifying and quantifying nucleic acid sequence aberrations using hybridization probes.
2. Description of Related Art
Hybridization probes are widely used to detect and/or quantify the presence of a particular nucleic acid sequence within a sample of nucleic acid sequences. Hybridization probes detect the presence of a particular nucleic acid sequence, referred to herein as a target sequence, through the use of a complimentary nucleic acid sequence which selectively hybridizes to the target nucleic acid sequence. In order for a hybridization probe to hybridize to a target sequence, the hybridization probe must contain a nucleic acid sequence that is complementary to the target sequence. The complementary sequence must also be sufficiently long so that the probe exhibits selectivity for the target sequence over non-target sequences.
In order to design a hybridization probe that selectively hybridizes to a target sequence, one must first determine a nucleic acid sequence that is complementary to the target sequence. In applications where the target sequence is already known, for example, where one seeks to detect the insertion of a gene or promoter sequence into a vector or plasmid, a variety of methods are known for preparing highly selective hybridization probes. However, one limitation of hybridization assays is that one does not also know the target sequence in sufficient detail to prepare a selective hybridization probe.
Hybridization assays are most commonly designed to detect the presence or absence of a particular nucleic acid sequence, for example the insertion of a gene into a vector or plasmid. However, hybridization assays are generally not designed to detect the movement of a nucleic acid sequence relative to another nucleic acid sequences in a sample. The detection of nucleic acid sequence aberrations using a hybridization assay is limited by both the ability to design sequence specific probes and the ability to detect the movement of a nucleic acid sequence relative to other nucleic acid sequences in a sample. The detection of nucleic acid sequence aberrations is further limited by the infrequency of nucleic acid sequence aberrations. For example, chromosome translocations, a type of nucleic acid sequence aberration, is estimated to occur at a frequency on the order of 1 per 1,000,000 cells in a particular gene. Currently available hybridization assays are not able to accurately detect and quantify such infrequent genetic events. Although translocations are more frequent in the whole genome (approximately 1 per 200 cells), currently available assays are not practical for use in assaying the large number of individuals that must be evaluated in population studies.
As used herein, nucleic acid sequence aberrations refer to rearrangements between and within nucleic acids, particularly chromosomal rearrangements. Nucleic acid sequence aberrations also refer to the deletion of a nucleic acid sequence, particularly chromosome deletions. As used herein, the term "nucleic acids" refers to both DNA and RNA.
A chromosome translocation is an example of a nucleic acid sequence aberration. A chromosome translocation refers to the movement of a portion of one chromosome to another chromosome (inter-chromosome rearrangement) as well as the movement of a portion of a chromosome to a different location on that chromosome (intra-chromosome rearrangement). In general, chromosome translocations are characterized by the presence of a DNA sequence on a particular chromosome that is known to be native to a different chromosome or different portion of the same chromosome.
Chromosome translocations are frequently random genetic events which can occur at virtually any portion of any chromosome. Because the particular nucleic acid sequences involved in a chromosome translocation are not always known, it is generally not possible to design a hybridization probe that will uniquely identify a particular translocated sequence without first determining that translocated sequence. In addition, because chromosome translocations involve the movement of a nucleic acid sequence within a sample as opposed to the appearance or disappearance of the nucleic acid sequence, it generally is not possible to detect a chromosome translocation merely by assaying for the presence or absence of a particular nucleic acid sequence.
Chromosome translocations are known to be involved in carcinogenesis and inherited genetic disorders and have been shown to increase in frequency upon exposure to radiation and certain chemicals. Measurement of the frequency of chromosome translocations after exposure to radiation or a particular agent is therefore useful for evaluating the tendency of particular agents or forms of radiation to cause or increase the frequency of chromosome translocations.
Chromosome translocations are also known to be associated with specific diseases, including, for example lymphomas and leukemias, such as Burkitt's lymphoma, chronic myelocytic leukemia, chronic lymphocytic leukemia and granulocytic leukemia, as well as solid tumors such as malignant melanoma, prostate cancer and cervical cancer. A method for rapidly detecting a translocation associated with a disease is needed as a method for diagnosing disease.
Fluorescence in situ hybridization (FISH) using chromosome-specific composite hybridization probes ("chromosome painting") was developed as an assay for detecting chromosome translocations. FISH is described in Pinkel, et al., Proc. Natl. Acad. Sci. (USA) 83:2934-2938 (1986); Lucas, et al., International Journal of Radiation Biology 56:35-44 (1989), 62: 53-63 (1992); Pinkel, et al., Proc. Natl. Acad. Sci. (USA) 85:9138-9142 (1988), each of which is incorporated herein by reference.
The fluorescent hybridization probes used in FISH-based chromosome painting are chromosome-specific but not unique, i.e., they hybridize primarily to a particular chromosome type. Chromosome translocations are identified in the FISH assay by visually scanning individual cells for the presence of two different fluorescent signals on a single chromosome, the two fluorescent signals originating from two different FISH probes, each probe having homology to a different chromosome type.
Because each FISH probe hybridizes to a specific chromosome type and not to the chromosome translocation itself, it is not possible to determine the frequency of chromosome translocations directly from the fluorescence signal emanating from a FISH probe. Rather, the frequency of random chromosome translocations in a cell sample must be determined according to FISH assays by visually scanning individual metaphase cells or slides. The need to visually scan such individual cells effectively limits the number of cells that can be assayed, thereby reducing the sensitivity of the FISH assay and introducing the possibility of human error.
Accordingly, a fast, accurate method is needed for quantifying chromosome translocations and other nucleic acid sequence aberrations. In particular, a method is needed which can analyze the nucleic acids contained in a sample of cells for the presence of a nucleic acid sequence aberration without the need to analyze each cell individually.